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Methods. 2016 Sep 1;107:10-22. doi: 10.1016/j.ymeth.2016.03.006. Epub 2016 Mar 14.

Structural and functional assessment of APOBEC3G macromolecular complexes.

Author information

1
Department of Biochemistry and Biophysics, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA.
2
OyaGen, Inc, Rochester BioVenture Center, 77 Ridgeland Road, Rochester, NY 14623, USA.
3
Department of Biochemistry and Biophysics, University of Rochester, School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, NY 14642, USA; Wilmot Cancer Institute, 601 Elmwood Avenue, Rochester, NY 14642, USA; Center for RNA Biology, 601 Elmwood Avenue, Rochester, NY 14642, USA; OyaGen, Inc, Rochester BioVenture Center, 77 Ridgeland Road, Rochester, NY 14623, USA; Center for AIDS Research, 601 Elmwood Avenue, Rochester, NY 14642, USA. Electronic address: harold.smith@rochester.edu.

Abstract

There are eleven members in the human APOBEC family of proteins that are evolutionarily related through their zinc-dependent cytidine deaminase domains. The human APOBEC gene clusters arose on chromosome 6 and 22 through gene duplication and divergence to where current day APOBEC proteins are functionally diverse and broadly expressed in tissues. APOBEC serve enzymatic and non enzymatic functions in cells. In both cases, formation of higher-order structures driven by APOBEC protein-protein interactions and binding to RNA and/or single stranded DNA are integral to their function. In some circumstances, these interactions are regulatory and modulate APOBEC activities. We are just beginning to understand how macromolecular interactions drive processes such as APOBEC subcellular compartmentalization, formation of holoenzyme complexes, gene targeting, foreign DNA restriction, anti-retroviral activity, formation of ribonucleoprotein particles and APOBEC degradation. Protein-protein and protein-nucleic acid cross-linking methods coupled with mass spectrometry, electrophoretic mobility shift assays, glycerol gradient sedimentation, fluorescence anisotropy and APOBEC deaminase assays are enabling mapping of interacting surfaces that are essential for these functions. The goal of this methods review is through example of our research on APOBEC3G, describe the application of cross-linking methods to characterize and quantify macromolecular interactions and their functional implications. Given the homology in structure and function, it is proposed that these methods will be generally applicable to the discovery process for other APOBEC and RNA and DNA editing and modifying proteins.

PMID:
26988126
PMCID:
PMC5014647
DOI:
10.1016/j.ymeth.2016.03.006
[Indexed for MEDLINE]
Free PMC Article

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